1. Field of the Invention
This invention relates to a fluorescence analysis apparatus for detecting a specific substance, which is contained in a sample, by use of a fluorometric analysis technique. This invention particularly relates to a fluorescence analysis apparatus of a type, in which a sensor section is dipped in a liquid sample.
2. Description of the Related Art
Heretofore, in fields of biological analyses, and the like, a fluorometric analysis technique has been used widely as an analysis technique, which has a high sensitivity and is easy to perform. The fluorometric analysis technique is the technique, wherein exciting light having a specific wavelength is irradiated to a sample expected to contain a substance to be analyzed, which substance is capable of producing fluorescence by being excited by the exciting light having the specific wavelength, wherein the fluorescence having thus been produced by the substance to be analyzed is detected, and wherein the presence of the substance to be analyzed is thereby confirmed. In cases where the substance to be analyzed is not a fluorescent substance, a technique has heretofore been conducted widely, wherein a specific binding substance, which has been labeled with a fluorescent substance and is capable of undergoing the specific binding with the substance to be analyzed, is brought into contact with the sample, wherein the fluorescence is detected in the same manner as that described above, and wherein the occurrence of the specific binding, i.e. the presence of the substance to be analyzed, is thereby confirmed.
FIG. 13 is a schematic side view showing an example of a conventional fluorescence analysis apparatus for carrying out a fluorometric analysis technique utilizing a labeled specific binding substance. By way of example, the fluorescence analysis apparatus illustrated in FIG. 13 is utilized for detecting an antigen 2, which is contained in a sample 1. The fluorescence analysis apparatus illustrated in FIG. 13 comprises a base plate 3, on which a primary antibody 4 capable of undergoing the specific binding with the antigen 2 has been coated. The fluorescence analysis apparatus also comprises a sample support section 5, which is formed on the base plate 3. The sample 1 is caused to flow within the sample support section 5. A secondary antibody 6, which has been labeled with a fluorescent substance 10 and is capable of undergoing the specific binding with the antigen 2, is then caused to flow within the sample support section 5. Thereafter, exciting light 8 is irradiated from a light source 7 toward a surface area of the base plate 3. Also, an operation for detecting the fluorescence is performed by a photodetector 9. In cases where the predetermined fluorescence is detected by the photodetector 9, the specific binding of the secondary antibody 6 and the antigen 2 with each other, i.e. the presence of the antigen 2 in the sample, is capable of being confirmed.
In the example described above, the substance whose presence is actually confirmed with the fluorescence detecting operation is the secondary antibody 6. If the secondary antibody 6 does not undergo the specific binding with the antigen 2, the secondary antibody 6 will be carried away and will not be present on the base plate 3. Therefore, in cases where the presence of the secondary antibody 6 on the base plate 3 is detected, the presence of the antigen 2, which is the substance to be analyzed, is capable of being confirmed indirectly.
Particularly, with the rapid advances made in enhancement of performance of photodetectors, such as the advances made in cooled CCD image sensors, in recent years, the fluorometric analysis technique described above has become the means essential for biological studies. The fluorometric analysis technique has also been used widely in fields other than the biological studies. In particular, with respect to the visible region, as in the cases of FITC (fluorescence wavelength: 525 nm, quantum yield: 0.6), Cy5 (fluorescence wavelength: 680 nm, quantum yield: 0.3), and the like, fluorescent dyes having high quantum yields exceeding 0.2, which serves as a criterion for use in practice, have been developed. It is thus expected that the fields of the application of the fluorometric analysis technique will become wide even further.
Also, a fluorometric analysis technique utilizing an evanescent wave has heretofore been proposed. FIG. 14 is a schematic side view showing an example of a conventional fluorescence analysis apparatus for carrying out a fluorometric analysis technique utilizing an evanescent wave. In FIG. 14, similar elements are numbered with the same reference numerals with respect to FIG. 13.
In the fluorescence analysis apparatus illustrated in FIG. 14, in lieu of the base plate 3 described above, a prism (a dielectric material block) 13 is utilized. A metal film 20 has been formed on a surface of the prism 13. Also, the exciting light 8 having been produced by the light source 7 is irradiated through the prism 13 under the conditions such that the exciting light 8 may be totally reflected from the interface between the prism 13 and the metal film 20. With the constitution of the fluorescence analysis apparatus illustrated in FIG. 14, at the time at which the exciting light 8 is totally reflected from the interface described above, an evanescent wave 11 oozes out to the region in the vicinity of the interface described above, and the secondary antibody 6 is excited by the evanescent wave 11. Also, the fluorescence detecting operation is performed by the photodetector 9 located on the side of the sample 1, which side is opposite to the side of the prism 13. (In the cases of FIG. 14, the photodetector 9 is located on the upper side.)
With the fluorescence analysis apparatus illustrated in FIG. 14, the exciting light 8 impinges upon the aforesaid interface from below in FIG. 14 and at an angle such that the exciting light 8 may be totally reflected from the aforesaid interface. As a result, the evanescent wave 11, which is capable of reaching only a region of several hundreds of nanometers from the aforesaid interface, arises and excites the secondary antibody 6. Therefore, it is possible to minimize the occurrence of the problems in that the exciting light, which has been reflected and scattered by the liquid sample 1, and the fluorescence (self-fluorescence), which has been produced from the liquid sample 1 and the vessel having been excited by the exciting light 8, impinge upon the photodetector 9 and constitute the background with respect to the fluorescence signal to be detected. Accordingly, the evanescent fluorometric analysis technique described above has attracted particular attention for serving as a technique, which is capable of markedly suppressing (light) noise than with the conventional fluorometric analysis techniques, and with which the substance to be analyzed is capable of being fluorometrically analyzed in units of one molecule.
The fluorescence analysis apparatus illustrated in FIG. 14 is the surface plasmon enhanced fluorescence analysis apparatus, which has the sensitivity having been enhanced markedly among the fluorescence analysis apparatuses utilizing the evanescent fluorometric analysis technique. With the surface plasmon enhanced fluorescence analysis apparatus, wherein the metal film 20 is formed, at the time at which the exciting light 8 is irradiated through the prism 13, the surface plasmon arises in the metal film 20, and the fluorescence is amplified by the electric field amplifying effect of the surface plasmon. A certain simulation has revealed that the fluorescence intensity in the cases described above is amplified by a factor of approximately 1,000. The surface plasmon enhanced fluorescence analysis apparatus of the type described above is described in, for example, Japanese Patent Application Publication No. 10 (1998)-078390.
Also, as described in, for example, U.S. Pat. No. 4,703,182, as one of the apparatuses for carrying out the fluorometric analysis technique as described above, there has been known a fluorescence analysis apparatus comprising: (i) a light source for producing exciting light, (ii) a sensor section, which may be constituted of a rod-shaped glass section, or the like, the sensor section propagating the exciting light through the interior of the sensor section, the sensor section radiating out the thus propagated exciting light from an outside surface of the sensor section, such that the exciting light having thus been radiated out may excite a fluorescent substance for indicating the presence of a substance to be analyzed in a liquid sample, and (iii) a photodetector for detecting the fluorescence, which has been produced by the fluorescent substance when the fluorescent substance is excited by the exciting light. In cases where the analysis is to be performed by use of the aforesaid type of the fluorescence analysis apparatus, ordinarily, the sensor section is dipped in the liquid sample, and the fluorescent substance is excited by the exciting light, which has been radiated out from the sensor section into the liquid sample. Also, the fluorescence, which has been produced by the fluorescent substance having thus been excited by the exciting light, is detected by the photodetector.
In, for example, U.S. Pat. No. 4,582,809 and Japanese Unexamined Patent Publication No. 2006-047250, it is indicated that an optical fiber may be employed as the sensor section described above. Particularly, in, for example, U.S. Pat. No. 4,582,809, it is described that a fluorescent substance may be excited by an evanescent wave, which oozes out from the surface of the optical fiber.
The fluorescence analysis apparatus utilizing the sensor section, which is dipped in the liquid sample in the manner described above, has the advantages over a fluorescence analysis apparatus of a type, in which the sensor section is built in a part of a liquid vessel, and in which the liquid sample is introduced into the liquid vessel by use of a pump, or the like, in that the constitution of the fluorescence analysis apparatus is capable of being kept simple, and in that the cost of the fluorescence analysis apparatus is capable of being kept low.
However, with the conventional fluorescence analysis apparatus utilizing the sensor section, which is dipped in the liquid sample, the problems are encountered in that, in cases where an analysis is to be made with respect to a substance to be analyzed, the quantity of which is markedly small on the order of, for example, 1 pmol (picomol)/l (liter), a sufficient analysis accuracy is not capable of being obtained. The aforesaid problems are encountered markedly in cases where a colored liquid, such as whole blood, blood serum, or urine, which contains comparatively large quantities of absorbing and scattering constituents other than the substance to be analyzed, is the liquid sample, and in cases where the sensor section is constituted of a rod-shaped material made from glass, an integrally molded transparent resin, or the like, the cost of which is low, in lieu of being constituted of an optical fiber provided with a cladding layer or a covering layer. The aforesaid problems are caused to occur by a phenomenon wherein, while the exciting light and/or the received light is being propagated in the rod-shaped glass, or the like, which acts as an optical waveguide, the exciting light and/or the received light is brought into contact with the liquid sample at an interface, is thus scattered or absorbed, and is thereby attenuated. In order for the adverse effects of the aforesaid problems to be suppressed, ordinarily, the optical fiber provided with the cladding layer or the covering layer is often utilized as the sensor section. However, in the aforesaid fields, in which various articles are desired to be disposable, in cases where the aforesaid optical fiber is utilized as the sensor section, the cost of the expendable supplies is not capable of being kept low. Therefore, the sensor section constituted of the optical fiber is not capable of being employed for reasons of cost.
Also, in cases where a washing process for removing unnecessary constituents is provided, the rod-shaped sensor section, the cost of which is low, is capable of being employed. However, in such cases, a liquid transfer mechanism, such as a dispenser and a pump, the cost of which is high, becomes necessary for the washing operation, and the cost of the fluorescence analysis apparatus is not capable of being kept low.
Further, with the sensor section for making the fluorescence analysis in the state in which the sensor section is dipped in a liquid, such as the liquid sample, there is the risk that a reflectivity of the exciting light and/or the received light at the aforesaid interface and the total reflection conditions of an optical path, or the like, will alter in accordance with the technique for inserting the sensor section into the liquid, the quantity of the liquid, sway of the liquid, and the like. Therefore, with the sensor section for making the fluorescence analysis in the state in which the sensor section is dipped in the liquid, it is not always possible to keep good analysis reproducibility.